A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

Ahsan, Nagib; Huang, Yadong; Tovar‐Méndez, Alejandro; Swatek, Kirby N.; Zhang, Jingfen; Miernyk, Ján A.; Xu, Dong; Thelen, Jay J.

doi:10.1021/pr3009995

Cited by 28 publications

(47 citation statements)

References 40 publications

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“…KiC assay (11,12) data. Most of the data are from high-throughput experiments from different laboratories using different instrumentation and data mining strategies; therefore, the quality of the data varies among different studies.…”

Section: Methodsmentioning

confidence: 99%

P³DB 3.0: From plant phosphorylation sites to protein networks

Yao¹,

Ge²,

Wu³

et al. 2013

Nucl. Acids Res.

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In the past few years, the Plant Protein Phosphorylation Database (P3DB, http://p3db.org) has become one of the most significant in vivo data resources for studying plant phosphoproteomics. We have substantially updated P3DB with respect to format, new datasets and analytic tools. In the P3DB 3.0, there are altogether 47 923 phosphosites in 16 477 phosphoproteins curated across nine plant organisms from 32 studies, which have met our multiple quality standards for acquisition of in vivo phosphorylation site data. Centralized by these phosphorylation data, multiple related data and annotations are provided, including protein–protein interaction (PPI), gene ontology, protein tertiary structures, orthologous sequences, kinase/phosphatase classification and Kinase Client Assay (KiC Assay) data—all of which provides context for the phosphorylation event. In addition, P3DB 3.0 incorporates multiple network viewers for the above features, such as PPI network, kinase-substrate network, phosphatase-substrate network, and domain co-occurrence network to help study phosphorylation from a systems point of view. Furthermore, the new P3DB reflects a community-based design through which users can share datasets and automate data depository processes for publication purposes. Each of these new features supports the goal of making P3DB a comprehensive, systematic and interactive platform for phosphoproteomics research.

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Section: Methodsmentioning

confidence: 99%

P³DB 3.0: From plant phosphorylation sites to protein networks

Yao¹,

Ge²,

Wu³

et al. 2013

Nucl. Acids Res.

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“…First, functional protein microarrays containing 15,000 proteins were probed with purified ILK1-V5 and anti-V5 Cy3-labeled antibody, allowing identification of in vitro protein-ILK1 complexes . Second, in vitro phosphorylation targets ILK1 were identified using in a kinase client assay (KiC) where purified ILK1-cMyc was incubated with a peptide library and phosphorylation of peptides was quantified by mass spectrometry (Ahsan et al, 2013). These approaches revealed a number of putative ILK1 interactors including several transporters (Supplemental Table S3).…”

Section: Ion Accumulation Changes After Flg22 Treatmentmentioning

confidence: 99%

“…The kinase assay was conducted in kinase buffer (20 mM HEPES-KOH pH 7.4, 5 mM MgCl 2 or MnCl 2 , 1 mM DTT, 2 mM ATP; Ahsan et al, 2013). The kinase client assay was performed with 5 mM MnCl 2 with purified ILK1 and a mixture of approximately 210 synthetic peptides (8 mM) per sample from a library of 2095 peptides that were designed based on the in vivo phosphoproteomic dataset available on the P 3 DB Web site (http://www.p3db.org/).…”

Section: Mass Spectrometry Analysis and Kinase Client Assaymentioning

confidence: 99%

The Raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for Innate Immunity and Abiotic Stress Response

et al. 2016

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Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca 2+ uptake in signaling, the regulation and significance of PAMPinduced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H + )/K + symporter that mediates a high-affinity uptake during K + deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K + efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K + loss. Taken together, our results position ILK1 as a link between plant defense pathways and K + homeostasis.

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“…This result is consistent with the observation by Ahsan et al . () that AtPPI‐2 Ser‐140 can be phosphorylated by three different kinases.…”

Section: Resultsmentioning

confidence: 95%

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

et al. 2017

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Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we utilized a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor-like kinases. The symbiotic receptor-like kinases Nodulation Receptor-like Kinase (NORK) and Lysin motif domain-containing receptor-like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor-like kinases, very little is known about their phosphorylation substrates. Using this high-throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor-like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself which was also confirmed by pair-wise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high-throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.

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A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

Cited by 28 publications

References 40 publications

P³DB 3.0: From plant phosphorylation sites to protein networks

P³DB 3.0: From plant phosphorylation sites to protein networks

The Raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for Innate Immunity and Abiotic Stress Response

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

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A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

Cited by 28 publications

References 40 publications

P3DB 3.0: From plant phosphorylation sites to protein networks

P3DB 3.0: From plant phosphorylation sites to protein networks

The Raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for Innate Immunity and Abiotic Stress Response

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

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P³DB 3.0: From plant phosphorylation sites to protein networks

P³DB 3.0: From plant phosphorylation sites to protein networks